JOURNAL OF NEUROBIOLOGY, VOL.

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ISOLATION AND POLYMERIZATION OF BRAIN ACTIN STEPHEN MORING, MARY RUSCHA, P E T E R COOKE, and F R E D SAMSON Department of Physiology & Cell Biology, University of Kansas, Lawrence, Kansas 66045; and Ralph L. Smith Mental Retardation Research Center, University of Kansas Medical Center, Kansas City, Kansas 66103 SUMMARY

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isolating muscle actin are applied to brain, many different proteins are extracted. Tubulin, a major protein in brain with properties similar t o actin, is the major constituent. A method is described for isolating the “brain actin” to a purity of 90-95%. The isolation method begins with an extraction of bovine brain in low ionic strength buffer with ATP and sucrose. The extract is treated with NH4S04,MgC1, and KC1 and incubated at 37°C. A precipitate is formed which contains primarily tubulin and brain actin. Resolubilization of the brain actin is achieved with a low ionic strength buffer solution with sucrose and ATP. Further purification is accomplished by a cycle of polymerization-depolymerization. This “brain actin” shares with muscle actin the following properties: ( I ) Similar molecular weight and molecular charge as determined by SDS polyacrylamide gel and ordinary disc electrophoresis; (2) Polymerization to a filamentous form under the same conditions; (3) Contains 3-methylhistidine; ( 4 ) Vinblastine sulfate will induce filament formation. Actin-like protein has been identified in nonmuscular tissues including kidney, adrenal gland and brain (Puszkin, Berl, Puszkin and Clarke, 1968; Berl and Puszkin, 1970; Fine, 1971; Fine and Bray, 1971; Puszkin and Berl, 1972). Striking similarities have been described between microtubule protein, tubulin, and actin (Renaud, Rowe and Gibbons, 1968; Stephens and Link, 1969; Stephens, 1970). Berl and Puszkin (1970) identified and characterized an actin-like protein and a myosin-like protein in bovine brain, and named them neurin and stenin, respectively. They report that these proteins can be identified by superprecipitation, Sephadex chromatography, and sucrose gradient centrifugation. They have also reported that ( a ) neurin stimulates the Mg2+ ATPase activity of muscle myosin, ( b ) neurin and stenin behave analogous to actin and myosin in forming a protein complex as observed in muscle, ( c ) neurin binds ATP (0.5 mo1/50,000 g of protein) 245 @ 1975 by John Wiley & Sons, Inc.

MORING ET AL. and undergoes nucleotide exchange with I4C-ATP,and ( d ) neurin contains 3-methyl-histidine, a characteristic amino acid of muscle actin. I n this paper, we have explored various methods of extracting and isolating an actin-like protein from the brain, and an attempt has been made to contrast the electrophoretic, isoelectric, and aggregation properties of brain actin with the related proteins, muscle actin, and brain tubulin. METHODS

Preparation o f actin-like protein (neurin) from brain Neurostenin was prepared by the method of Puszkin and Berl (1972). Fresh bovine brain was obtained within 1 hr of slaughter, chilled on ice, cleaned, minced, and washed in 0.8% saline solution. The minced brain was homogenized in Weber-Edsall Extraction solution: 0.6 M KC1 in 0.01 M sodium carbonate buffer (pH 9.2) and kept a t 4°C for 16 hr. The homogenate was then centrifuged at 60,000 X g for 1 hr and the pellet was discarded. The neurostenin was precipitated from the supernatant fluid by diluting the KCl concentration to 0.1 M and reducing the pH to 6.3 with 0.125 M acetate buffer (pH 4.9). The precipitate was sedimented by centrifugation a t 20,000 X g for 10 min and solubilized in 0.05 M Tris-0.6 M KCl (pH 7.2), and reprecipitated by repeating the above. The product, “2 cycle neurostenin,” was then dissolved in 0.05 M Tris-0.6 M K I - 1 m M ATP a t pH 7.8 and applied to a calibrated Sephadex G-200 column (2.5 cm X 45 mm). Fractions were collected from the column in 2 ml aliquots and assayed for relative protein concentration by measuring the absorbance a t 280 n m on a Beckman spectrophotometer.

Preparation of tubul in by uinblastine precipitation Ten grams of brain was homogenized in an equal amount (w/v) of a solution containing 0.01 M potassium phosphate (pH 6.5), 0.01 M magnesium chloride, and 0.24 M sucrose (PMS buffer), centrifuged at 48,000 X g for 20 min, and the sediment discarded. The extract was then centrifuged again a t 100,000 X g for 1 hr, after which the sediment was discarded and the supernatant fluid adjusted to 0.2 m M with 2 m M vinblastine sulfate and incubated a t 37°C for 1 hr. The precipitate which formed was sedimented by centrifugation a t 100,000 x g for 1 hr, and resuspended in a small volume of 0.01 M potassium phosphate-0.01 M magnesium chloride buffer (pH 6.6).

Preparation of muscle actin G-actin was extracted and isolated from an acetone powder prepared from the hind quarters of a rabbit by the Straub procedure as modified by Szent-Gyorgyi (1945). The acetone powder was extracted in a solution of 0.5 m M ATP, 0.5 m M 2-mercaptoethanol, and 0.2 m M CaC12 a t 4°C for 1 hr. The supernatant fluid from the extract was adjusted to 0.6 M KCl and 1 m M MgCl,, and allowed to stand at 20°C for 2 hr. The polymerized F-actin was sedimented by centrifugation a t 100,000 X g for 2 hr. F-actin was then depolymerized in the extraction solution, and dialyzed against the same solution for 16 hr. The fraction was centrifuged again a t 100,000 X g for 2 hr to sediment the impurities. The G-actin was further purified by gel chromatography on a Sephadex G-200 column according to Rees and Young (1967).

Sephadex chromatography A 2.5 cm X 45 cm column was poured with G-200 fine Sephadex gel and equilibrated at reverse flow with 0.5 m M ATP-0.5 m M 2-mercaptoethanol 0.2 m M CaCIZ (pH

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7.2-7.8). The column was allowed to flow a t the rate of 6 ml/hr a t 4°C. Ten milliliters of protein a t a concentration between 5 and 10 mg,’ml was applied to the column through a n open syringe.

Protein determination Protein concentration was determined by Lowry assay (Lowry, Rosebrough, Farr, and Randall, 1951). The standard curve was determined by serial dilution of a standard solution of bovine serum albumin from Sigma Chemical Company.

S D S-polyacryl am ide electrophoresis T h e protein constituency and protein molecular weights were determined by SDSpolyacrylamide gel electrophoresis as described by Weber and Osborn (1969). Protein samples, between 2-5 mg/ml concentration, were incubated a t 37OC for 2 -16 hr, and mixed with a drop of tracking dye and a drop of glycerol and applied directly to each gel.

Disc electrophoresis Standard gel electrophoresis was performed a t room temperature on 6.8% polyacrylamide gel containing 8 M urea with 0.12 M 2-mercaptoethanol. A current of 3-4 mA/gel was applied and run continuously until the tracking dye passed to the bottom of the gels. The buffer was 0.025 A4 Tris -0.19 M glycine (pH 8.2). The gels were fixed in 10% TCA for 30 min and stained overnight in 0.2% aqueous Coomassie blue. Destaining was carried out in 7 % glacial acetic acid.

Colchicine binding assay Assay of colchicine binding on neurostenin fractions was carried out according to the met’iod of Weisenberg, Borisy and Taylor (1968). The protein fractions of different concentrations were prepared ’on a volume to volume basis, with a final incubation volume of 0.5 ml. All other procedures were carried out as previously described.

Materials Nae ATP, grade 11, was obtained from Sigma Chemicals. Vinblastine sulfate was donated by the Lilly Laboratories (purity 88.3%). Sephadex G-200 medium and fine were purchased from Pharmacia Company. Acrylamide was obtained from Eastman Chemicals. 3H-colchicine was obtained from New England Nuclear Company. All other reagents used were of an analytical grade quality. RESULTS

Neurostenin was prepared from bovine brain and rabbit brain by the method of Puszkin and Her1 (1972). Assay by SDS-electrophoresis of 2 cycle neurostenin showed that tubulin (mol wt 52,000-55,000) is the major constituent polypeptide (Fig. 1). Other small amounts of protein in the fraction consisted of three high molecular weight proteins (between 150,000--2OO,OOO), one a t a molecular weight of 38,000, and neurin (mol wt, 45,000). The presence of tubulin in the preparations of neurostenin was confirmed by their binding with. 3H-colchicine. A one cycle neurostenin fraction (first step precipitation of neurostenin) was prepared by extracting the proteins from the homogenate of brain for 2 hr instead of 16 hr. The extraction and isolation procedure was shortened due t o the

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Fig. 1. SDS-acrylamide electrophoresis of 1 cycle neurostenin (A); 2 cycle neurostenin (B); vinblastine-precipitate of protein from bovine brain (C); supernatant fraction of soluble protein from bovine brain (D); G-actin from bovine muscle by method of Rees and Young (1967) (E); neurin isolated by precipitation with (NH4),S04, MgC1, and KCl (F); neurin purified by 2 stel) polymerization-depolymerization (G). Tubulin (T),neurin (N).

rapid decay of colchicine binding activity of tubulin. The one cycle neurostenin fraction assayed with 3H-colchicine (5 Ci/mM) produced an activity of 55,000 cpm/mg of protein. One cycle neurostenin was found to contain essentially the same polypeptides as the two cycle fraction (Fig. 1). Numerous attempts to isolate neurin from the 2 cycle fraction of “neurostenin” on a Sephadex G-200 column were unsuccessful. Assay of fractions collected in the void volume and the retarded volume showed essentially the same band patterns on SDS gels. I n the majority of cases, most of the neurin and tubulin were eluted in the void volume (Fig. 2).

Vinblastine precipitate of soluble fraction of brain Vinblastine precipitation of the soluble fraction of bovine brain isolated three major proteins. When the solubilized precipitate was applied to SDS gels, a 52,000-55,000 molecular weight polypeptide, presumably tubulin; a 45,000 molecular weight protein, presumably neurin; and an

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Fig. 2. Column chromatography of bovine neurostenin and bovine muscle G-actin. G-200 columns were equilibrated and eluted on reverse flow with 0.6 M KI-1 mM ATP-0.05 M Tris (yII 7.6) and 0.5 mM ATP-0.5 m M 2-mercaptoethanol-0.2 mM CaCl,, respectively. Arrows indicate elution position of blue dextran (B.D.) and G-actin. (0)Elution profile for neurostenin; ( 0 ) elution profile for G-actin. Protein concentration measured by absorbance a t 280 nm.

unknown protein of 38,000 molecular weight were found. Based on the stain intensity of protein on the gels, the percentage of neurin and tubulin to the total protein in the sample was estimated spectrophotometrically to be 20% and 40%, respectively. The yield of neurin by vinblastine precipitation is substantially greater than that observed in the Puszkin and Berl (1972) preparation (Fig. 1).

Extraction and isoLation of neurin from bovine bruin A procedure was developed in an attempt t o improve the purity of preparations of the actin-like protein from brain. The method involves preparing an extract of the soluble fraction of brain utilizing a low ionic strength buffer solution, and salting out and precipitation of soluble protein with the use of ammonium sulfate, magnesium chloride, potassium chloride, and incubation a t 37°C. Preparations were made from frozen brain, which was obtained within 1 hr after slaughter. The brains were cleaned of meningies, minced and washed in 0.8% saline solution, and frozen and stored a t -40°C for later use. One hundred grams of frozen brain was thawed and washed once in 3 volumes of 0.8% saline solution. The brain mince was homogenized with a glass-Teflon homogenizer in an equivalent volume of PMS buffer (previously described) containing 1 m M of ATP. The homogenate was allowed to extract for 30 min a t 4"C, and was centrifuged a t 50,000 X g for 20 min. The sediment was discarded. The extract was then cen-

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trifuged a t 100,000 X g for 1hr, and the sediment discarded. The supernatant fluid was adjusted to 10% with ammonium sulfate crystals, and allowed to stand a t 4°C for 15 min. The fraction was centrifuged a t 50,000 X g for 30 min, and the sediment discarded. The resultant Supernatant fraction was adjusted t o 0.1 M with crystals of KC1, and the pH was lowered to 5.8 with dilute HC1. The sample was then incubated a t 37°C for 1 hr. A rapid change in turbidity was noted, however no change in viscosity was observed a t the end of the period of incubation. A precipitate formed, and was sedimented by centrifugation at 30,000 X g for 30 min. The supernatant fluid was decanted and discarded. The precipitate was homogenized in 10 volumes (w/v) of a solution of 1 m M ATP, 0.5 m M 2-mercaptoethanol, 0.2 m M MgC12, 0.24 M sucrose, and 5 m M Tris-HC1, pH 7.4-7.8 (AMMST buffer) with two changes of the buffer. The sample was removed from dialysis bags, and the insoluble protein was sedimented by centrifugation at 100,000 X g for 1 hr. The supernatant fraction containing soluble protein was assayed by the Lowry method and by SDS-gel electrophoresis. This fraction was found t o be approximately 80% pure neurin, and the yield was determined to be from 15 to 20 mg of protein (Fig. 1F). Identification of the actin-like protein was made by comigration of the protein with rabbit muscle actin on SDS gels.

Polymerization of neurin isolated from brain Neurin, prepared by the method just described, could be induced to polymerize when in solution in AMMST buffer. When a sample of the isolated protein was adjusted to 0.1 M KC1 and 2 m M MgCL, and incubated for 1 hr a t 22”C, a moderate increase in viscosity was observed. A small sample of the protein was placed on a carbon-coated grid and stained with 2% uranyl acetate. Under the electron microscope, actinlike filaments of a diameter of approximately 60 were observed (Fig. 3). Using potassium and magnesium salts to induce polymerization, neurin could be further purified by sedimentation of filamentous neurin by centrifugation at 100,000 X g for 2 hr, and depolymerization of the “Fneurin” pellet by resuspension and dialysis with continuous stirring in the AMMST buffer for a period of 24 hr. The impurities were removed from the fraction of soluble neurin by centrifugation at 100,000 X g for 2 hr. The supernatant fraction was assayed by SDS electrophoresis and was found to be approximately 90-95% pure (Fig. 1G). The final yield using this method produced from 2 to 5 mg of the protein.

Polymerization of actin by vinblastine Since vinblastine was found to precipitate the actin-like protein in the brain, the effects of vinblastine on G-actin isolated from muscle and neurin isolated by our method were studied. Various concentrations of vinblastine ranging from 2 m M to 0.1 m M were added to fractions of G-actin

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Fig. 3. Electron micrograph of polymerized bovine brain actin-like protein. Neurin was purified by previous polymerization and depolymerization. The purified globular protein was adjusted to 0.1 M KC1 and incubated for 1 hr a t 20°C. Samples were placed on carbon-coated grids, and stained with 2 % uranyl acetate. (Magnification 119,200 X .)

(10 mg/ml, containing 0.5 m M ATP-0.5 mM 2-mercaptoethanol-0.2 m M CaC1,) and neurin (2 mg/ml in AMMST buffer) in one to two dilutions. The vinblastine treated fractions were incubated a t 4°C and 22°C for 1 hr. At 4"C, in samples containing 0.5 m M or higher concentration of vinblastine, the formation of a flocculent precipitate was observed immediately after addition of vinblastine. I n fractions incubated a t 22 "C, a rapid increase of viscosity was observed minutes after the vinblastine

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Fig. 4. Electron micrograph of polymerized rabbit muscle actin. G-actin was incubated in 1 m M vinblastine sulfate for 1 hr at 2OOC. Samples were placed on carbon-coated grids, and stained with 2% uranyl acetate. (Magnification 85,800 X .)

was added. Electron microscopy of the samples incubated a t 22°C revealed the presence of uniform sized aggregates of the protein, approximately 800 in diameter, and filaments, approximately 60 in diameter and about 1 micron in length (Fig. 4). Polymerization of G-actin and neurin in the presence of vinblastine is temperature dependent as well as being dependent on the final concentration of vinblastine in the protein solution. Concentrations below 0.5 m M were found to be ineffective in inducing polymerization.

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M

Fig. 5. Densitometer tracings of 7.5% acrylamide gels. A, B, C , SDS gels: (A) vinblastine precipitated protein from soluble fraction of brain, 10 pg protein, “T” tubulin, 54,000 mol wt and “N” neurin, 45,000 mol wt; (B) muscle G-actin purified on Sephadex G-200 column, 2 pg protein; (C) comigration of vinblastine precipitated protein from brain and muscle G-actin, 10 pg and 2 pg, respectively. (D and E) Urea-mercaptoethanol gels: (D) vinblastine precipitated protein from above, “T” tubulin, and “N” neurin; (E) comigration of vinblastine precipitated protein and muscle G-actin, “M” contaminating proteins.

Mobility of neurin and actin on polyacrylamide gels Neurostenin, prepared by the method of Puszkin and Berl, and the vinblastine precipitate of bovine brain protein were assayed with muscle G-actin by SDS-polyacrylamide and standard gel electrophoresis. Equal concentrations of either fraction containing neurin were added to samples of G-actin, and applied%o the same gel. Assay using both methods of electrophoresis revealed complete and symmetrical comigration, suggesting that neurin and actin share the same molecular weight and molecular charge properties (Fig. 5). DISCUSSION

This study confirms the earlier reports from others (Puszkin and Bed, 1968) that actin which is almost indistinguishable from muscle actin is present in brain. Further, it describes a procedure which yields a 9095% pure sample of the protein in a form that will polymerize to form typical actin filaments under conditions which induce filament formation with soluble actin. This brain actin has been named “neurin” by Puszkin and his colleagues (Puszkin et al., 1968), and this term is adopted here for the sake of convenience. The cellular source of this actin, that is, the relative amounts from neuronal versus glial type cells, is not known.

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A procedure for yielding pure preparations of the neurin was desirable because the method of Puszkin and Berl (1972) yields a small amount lof neurin (approximately 5 % of the protein isolated) and is approximately 80 % tubulin as determined by SDS electrophoresis and colchicine binding. Various methods were employed in an attempt t o isolate neurin free of tubulin and in adequate yield from the soluble fraction of brain. A t tempts t o isolate neurin from various types of extracts by precipitation with vinblastine, magnesium, and calcium were not selective, since other proteins were precipitated, including tubuiin. The behavior of neurin and tubulin were so similar under these conditions that it was not possible to solubilize one without the other. Even when these proteins were extracted, as with the Guba-Straub preparation in muscle, gel filtration could not be used to separate neurin from tubulin. The method presented here yields neurin in a highly purified form, The brain proteins precipitated by 37°C incubation in the presence of ammonium, potassium, and magnesium salts are largely neurin and tubulin. The precipitated proteins are then dialyzed against the AMMST buffer which solubilizes the neurin. The neurin is then separated from the insoluble proteins by centrifugation. When ammonium sulfate was not present during incubation, the neurin remained insoluble, and thus could not be separated from the other proteins in the precipitate. Since neurin could be isolated in pure form, it was possible to assay the protein for the presence of 3-methylhistidine. This rare amino acid is known to be a constituent of muscle and nonmuscle actins (Johnson and Perry, 1968; Woolley, 1972). Amino acid analysis revealed that 3-methylhistidine was present in this preparation, as shown previously for the neurin preparation of Puszkin and Berl (1972). The relatively small amount of 3-methylhistidine is difficult to quantify because of the great abundance of other amino acid residues but was found t o be not less than 0.1 mol per mol of neurin. Certain conditions were found to be important for the polymerization of the neurin. I n the absence of sucrose the neurin failed to polymerize and appeared to denature within a few hours. Sucrose is known to favor the polymerization of muscle actin (Kasai, Nakano and Oosawa, 1965) and to favor polymerization of other proteins such as tobacco mosaic virus protein (Lauffer and Stevens, 1968). Also, the polymerization of neurin is inhibited by calcium. When calcium was present or had been substituted for magnesium in AMMST buffer, the degree of polymerization was markedly reduced. That is, with calcium present (0.2 mM) fewer actin-like filaments were observed and those that were observed were shorter in length. If calcium (2 mM) was used in addition t o 0.1 M KC1 to induce polymerization of neurin, little or no change in viscosity of the protein solution was observed in contrast to a threefold increase in viscosity when magnesium was used. Vinblastine sulfate induces the polymerization of neurin or of muscle actin to form the typical filaments. It has been suggested that vinblastine competes with calcium for binding sites on tubulin and actin (Weiscnberg and Timasheff, 1970; Nimni, 1972). However, this phenomenon indicates that the vinblastine interaction is different from that of calcium.

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The functions of actin in brain cells would be expected to be similar t o those in muscle, that is, an element in cliemomechanical energy coupling processes. Several known events in the nervous system clearly require some energy coupling system: axoplasmic transport, transmitter release, and motility (as in development or regeneration). What role actin may have in these phenomena remains to be seen.

This research was aided by a grant from National Institutes of Health N S 01151 and Biomedical Sciences Support Grant RR07037 to the University of Kansas. The authors acknowledge the technical assistance of Joseph Fix.

REFERENCES

BERL, S. and PUSZKIN, S. (1970). Mgz+-Ca”+-activated adenosine triphosphatase system isolated from mammalian brain. Biochem. 9, 2058. FINE, R. E. (1971). Heterogeneity of tubulin. Nature New Biol. 233, 283. FINE,R. E. and BRAY,D. (1971). Actin in growing nerve cells. Nature New Biol. 234, 115. JOHNSON, P. and PERRY,S. L. (1968). Chemical studies on the cysteine and terminal peptides in triptic digests of actin. Biochem. J . 110, 207. E., and OOSAWA, F. (1965). Polymerization of actin free from KASAI,M., NAKANO, nucleotides and divalent cations. Biochim. Biophys. Acta 94, 494. LAUFFER,M. A. and STEVENS,C. L. (1968). In: Advances in Virus Research, vol. 13, K. M. Smith and M. A. Lauffer, Eds., Academic Press, New York, pp. 1-63. 0. H., ROSERROUGH, N. J., FARR,A. L., and RANDALL, R. J. (1951). Protein LOWRY, measurement with the folin phenol reagent. J . Biol. Chem. 193, 265. NIMNI, M. E. (1972). Vinblastine sulfate: Its reversible thermal aggregation and interaction with hydrophobic groups. Biochem. Pharmacol. 21, 485. PUSZKIN,S., BERL, S., PUSZKIN, E., and CLARKE,D. B. (1968). Actomyosin-like protein isolated from mammalian brain. Sczence 161, 170. PUSZKIN,S. and BERL,S. (1972). Actomyosin-like protein from brain: Separation and characterization of the actin-like component. Biochem. Biophys. Acta 256, 695. REES, M. K. and YOUNG,M. J. (1967). Studies on the isolation and molecular properties of homogeneous globular actin. J. Biol. Chem. 242, 4449. RENAUD,F. L., ROWE,A. J., and GIBBONS,I. R. (1968). Some properties of the proteins forming outer fibers of cilia. J. Cell Biol. 36, 79. STEPHENS, R. E. (1970). On the apparent homology of actin and tubulin. Science 168, 845. STEPHENS,R. E. and LINK, R. J. (1969). A comparison of muscle actin and ciliary microtubule protein in the mollusk (Pecten irradians). J. Molec. Biol. 40, 497. SZENT-GYORGYI, A. (1945). Studies on muscle. Acta Physiol. Scand. 9 (suppl.), 87. WEBER,K. and OSBORN,M. J. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J . Biol. Chem. 244, 4406. WEISENBERG,R. C., BORISY,G. G., and TAYLOR, E. W. (1968). The colchicinebinding protein of mammalian brain and its relation to microtubules. Biochem. 7, 4466. WEISENBERG, R. C. and TIMASHEFF, S. N. (1970). Aggregation of microtubule subunit protein. Effects of divalent cations, colchicine and vinblastine. Biochemistry 9, 4110. WOOLLEY,D. E. (1972). An actin-like protein from amoebae of Dictyostelium discoideum. Biochem. Biophys. 150, 519.

Accepted for publication September 11, 1974

Isolation and polymerization of brain actin.

The studies presented here confirm earlier reports that an actin-like protein is abundant in brain. However, when the traditional procedures for isola...
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